Investigations of the biosynthesis of two novel microbial metabolites will be continued. The first is the antitumor agent valanimycin, an azoxy compound produced by Streptomyces viridifaciens. As a naturally occurring azoxy compound, valanimycin is a member of a growing family of natural products that includes substances with antitumor, antifungal, and carcinogenic activity. Previous investigations have led to the elucidation of the early steps in the valanimycin biosynthetic pathway and, more recently, to the cloning and analysis of the valanimycin biosynthetic gene cluster. The long term objective of future studies of valanimycin is to identify and investigate the proteins encoded by the gene cluster that are responsible for the formation of the azoxy group of valanimycin, and thereby to elucidate the biochemical mechanism of azoxy group formation. These studies are significant because they should help illuminate the chemistry of N-N bond formation associated with the biosynthesis of other known bioactive natural products that contain N-N bonds. Future studies of valanimycin have several specific goals. The first goal is to investigate the role played by VImL, an apparent seryl-tRNA synthetase, in valanimycin biosynthesis, including an investigation of the possibility that a seryl-tRNA is an intermediate in the valanimycin biosynthetic pathway. The second goal will be to overproduce and investigate the function of several other proteins in the gene cluster that may be involved in the processing of the known intermediate isobutylhydroxylamine and in the processing of a serine derivative such as seryl adenylate or a seryl-tRNA. The third goal will be to create non-polar disruptions in genes of unknown function in the gene cluster and analyze the chemical phenotype of the resulting mutants to detect new intermediates in the pathway. The second metabolite to be investigated is the antitumor agent sparsomycin, a potent inhibitor of protein biosynthesis produced by S. sparsogenes. Sparsomycin is an important target for biosynthetic investigation because it is known to inhibit the peptidyltransferase step in protein biosynthesis and because previous studies have shown that sparsomycin biosynthesis involves novel biochemistry. Future investigations of sparsomycin will focus on cloning the sparsomycin resistance gene from S. sparsogenes to understand the mechanism of self-resistance in this organism and to gain access to the biosynthetic genes.